Screening Of Anti-influenza Virus Natural Products And Assessment

Influenza virus, the largest genus of Orthomyxoviridae family, based on the virus particle nucleoprotein (NP) and membrane protein (MP) antigen characteristics and genetic characteristics of different influenza viruses are divided into A, B, C. Influenza A virus in accordance with its surface antigen (H and N) and its genetic characteristics can be divided into many different subtypes. It has been found 15 known hemagglutinin subtypes (H1~15) and neural 9 NA subtypes (N1~9). The distinctive feature of influenza is high incidence, low mortality rate. But the total number of deaths caused by it is considerable. And as a result of influenza virus antigen, particularly the HA protein, which is mutate constantly, not only hard to the preparation the vaccines so that the effect of vaccine immunization will decline quickly, or even invalid. Until now, there are no influenza virus specific drugs available for clinical use, which is the same as the other viral diseases. Thus, new and more effective antiviral agents for future therapy in influenza virus infection are desired. In this study, we will screen the antiviral natural products against influenza virus A/PR/8/34(H1N1) in vitro and in vivo model and separate active site, for the aim of supplying references to the discovering new antiviral drugs.In the first part of the study, we established a rapid and reliable method for screening potential antiviral agents against influenza virus base on the cytopathogenic effect inhibition assay; MTT-based colorimetric assay and the hemagglutination assay. We have detected the anti-influenza virus A/PR/8/34(H1N1) activity of 160 natural products, including effect on viral replication, effect on viral adsorption and subsequent replication, and viral inactivation. The results showed that there were 21 aqueous extracts exhibited anti-influenza virus A/PR/8/34(H1N1) by the three different antiviral assays. Among of them, the aqueous extract from Spatholobus suberectus Dunn. showed potent antiviral activity against A/PR/8/34(H1N1) with its IC50 was 74.5μg/mL and TI was 13.3.In order to evaluate the effective of the aqueous extract from Spatholobus suberectus Dunn. inhibiting the influenza virus , we designed different assays in vitro and in vivo. In vitro: we determined the antiviral effects against three types of influenza virus on MDCK cells, which result show that the aqueous extract from Spatholobus suberectus Dunn.could inhibit all three virus. And the directly hemagglutinin assays and the reproduction of chick embryo assays showed that the A/PR/8/34 (H1N1) virus could be inactivation by the aqueous extract from Spatholobus suberectus Dunn. In vivo: The aqueous extract of Spatholobus suberectus Dunn. on acute toxicity in Kunming mice showed the oral LD50 is 800.49±38.75mg/kg. In the use of A/PR/8/34 (H1N1) virus strains of the establishment of appropriate mouse pneumonia virus mouse model based on a series of experiments. Experimental results show that the aqueous extract from Spatholobus suberectus Dunn. perfusion were able to significantly reduce the 5d and 9d mouse mortality in mice infected with a virus titer of lung tissue and necrosis in mouse lung slices and the level of infiltration and to inhibit A / PR/8/34 (H1N1) virus replication in lung tissue of mice. Description the aqueous extract from Spatholobus suberectus Dunn. could significantly prolong the survival time of infected mice, the protection of infected lung tissue.In order to investigate the mechanism of the aqueous extract from Spatholobus suberectus Dunn. inhibiting the infection of A/PR/8/34 (H1N1) we designed three different assays. Firstly, a assays design to investigate the time course effect of the virus infection at different concentration of the aqueous extract of Spatholobus suberectus Dunn. revealed it can inactivated A/PR/8/34 (H1N1) in vitro. Secondly the RT-PCR results showed that: the aqueous extract from Spatholobus suberectus Dunn. could inhibit the A/PR/8/34 (H1N1) virus replication. Effected of the aqueous extrat from Spatholobus suberectus Dunn. on immune organ of mice is assayed, the results showed that immune organ coefficient, such as thymus and spleen, augment obviously, burst coefficient is significant compared with control group. In vitro, the aqueous extract from Spatholobus suberectus Dunn. can enhance proliferation of control group and extract-hemolymph T cell stimulated by Con A. At the same time, it could promote proliferation clearly of T lymphocyte induced by Con A and B lymphocyte induced by LPS.The Spatholobus suberectus Dunn. antiviral properties were worth further investigated to identify the active sites. In summary, the active site was tracing by antiviral pharmacology assay in vitro. Firstly, the Spatholobus suberectus Dunn. powders were extracted with heated water. The extract was further purified by system extract assay, which used polyamide column chromatography methods. The results showed that the 50% -70% ethanol polyamide separation of the Spatholobus suberectus Dunn. parts were the active part. The characters of active site were identified by physics character experiments. The major ingredient of the alcohol was flavonoid, which were the most bio-active ingredient of the Spatholobus suberectus Dunn..In conclusion, the main results are as follows:1. Establishing a rapid and reliable method for screening potential antiviral agents against the influenza virus A / PR/8/34 (H1N1) and aquiring 21 aqueous anti- influenza virus A / PR/8/34 (H1N1) strongly.2. The aqueous extract from Spatholobus suberectus Dunn. could inhibit the influenza virus A / PR/8/34 (H1N1) in vitro and in vivo.3. The aqueous extract from Spatholobus suberectus Dunn. could inhibit influenza virus A / PR/8/34 (H1N1) RNA sysnthesis selectively and hence inhibit virus replication, also stimulated the immune organ coefficient and the proliferation of T lymphocyte and B lymphocyte.4. The aqueous extract from Spatholobus suberectus Dunn. could inhibit influenza virus A / PR/8/34 (H1N1) RNA sysnthesis selectively and hence inhibit virus replication, also stimulated the immune organ coefficient and the proliferation of T lymphocyte and B lymphocyte.5. The major anti-influenza virus A / PR/8/34 (H1N1) site of the Spatholobus suberectus Dunn. was mostly flavonoids.